title: neutrophils, Untreated

city: Tübingen

first: Marissa

last: Dubbelaar

address: Auf der Morgenstelle 15

department: Peptide-based Immunotherapy

institute: University and University Hospital Tübingen

data_processing:
The data was quantified using the Homo sapiens GRCh38 reference genome with Salmon (1.5.0)
Quantified data was loaded into R with tximport (1.30.0) and the TPM values were visualized with ggplot2 (3.4.4)
Assembly: GRCh38
Supplementary files format and content: Tab-delimited file containing the TPM values from all samples

source_name: Peripheral blood primary human neutrophil

extract_protocol:
Primay human neutrophils were isolated from freshly drawn healthy donor veinous blood upon obtaining informed consent, neutrophils isolated using standard Ficoll isolation, quality checked, and stimulated to induce the formation of NETs. Briefly, NETs were prepared by 600 nM PMA treatment for 4 h at 37°C and 5% CO2, or cells were left untreated during the incubation as the mock control. After incubation, the neutrophils were gently washed three times with PBS to get rid of PMA as the stimulus for NET formation, any cytokines released by the cells, unbound free RNA and unstimulated PMNs, as those do not adhere to the uncoated petri dish used here.  10 U/μL RNase inhibitor was added to the PMNs during NET formation. After the above-described washing process, PMA or mock NETs were resuspended in 300 μL of ML buffer and RNA isolation was performed according to the manufacturer’'s instructions (NucleoSpin miRNA isolation kit, Macherey-Nagel, 740971.50). As comparison total neutrophils were lysed as suggested in the kit manual. The RNA was eluted in 50 μL RNase DNase-free H2O and the concentration was determined with a Nanodrop Spectrophotometer. For isolation of whole PMN RNA, untreated PMNs were directly lysed, and the RNA was isolated from the cells according to the manufacturer’s instructions.  Isolated naRNA was analyzed for quality control using the Agilent 4200 TapeStation system. Subsequently, the RNA was sequenced according to NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® using the protocol for use with rRNA Depleted FFPE RNA.
NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® using the protocol for use with rRNA Depleted FFPE RNA

value: Untreated


title: neutrophils, 600nM PMA rep 5

status: Public on May 06 2024

type: SRA

country: Germany

library_source: transcriptomic

molecule: total RNA

organism: Homo sapiens

tag: cell type

value: Peripheral blood primary human neutrophil

tag: treatment

value: PMA


title: neutrophils, 600nM PMA rep 4


title: neutrophils, 600nM PMA rep 2


title: K1718-IDE-R-R1

city: Bellinzona

first: Luciano

last: Cascione

state: Ticino

address: Via Chiesa 5

department: Lymphoma & Genomics Research Program

country: Switzerland

institute: IOR

data_processing:
Illumina Casava1.7 software used for basecalling and demultiplexing.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome using STAR with default paramenters. Genes' Expression have been quantified using HT-Seq count using gencode v22 as gtf.
Read counts have been normalized with TMM algorithm, and converted into log2 counts per million of mapped reads (cpm)
Assembly: hg38
Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample (Raw_Counts sheet)
Supplementary files format and content: Matrix table with lcpm for every gene in every sample (Normalized lcpm)

source_name: cell line

extract_protocol:
Trizol reagent
Total RNA samples were prepared for RNA-Seq with the NEBNext rRNA Depletion kit, the NEBNext Ultra Directional RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina (New England BioLabs Inc.).

tag: condition

value: B-cell

tag: replicate

tag: phenotype

value: resistant to Idelalisib


title: neutrophils, 600nM PMA rep 3


title: neutrophils, 600nM PMA rep 1


title: K1718-IDE-R-R2


title: Sample 4_VL51-IDE-R-R2

data_processing:
Illumina Casava1.7 software used for basecalling. Four fastq files per samples.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to hg38 whole genome using STAR with default paramenters. Genes' Expression have been quantified using HT-Seq count using gencode v22 as gtf.
Genome_build: hg38
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

extract_protocol:
RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: cell line

value: Resistant


title: WT-GmMKK2 H. glycines infected RNA bio rep 2 [W102R3_2]

city: Knoxville

first: Tarek

last: Hewezi

state: TN

address: 2431 Joe Johnson Drive

department: Plant Sciences

country: USA

institute: University of Tennessee

data_processing:
Trimmomatic - fastq trimming and pairing
STAR - mapping the reads to a reference genome
Htseq - counting the reads mapped
DEseq2 - the differential expression analysis
Assembly: Gmax_508_v4.0
Supplementary files format and content: read counts

organism: Glycine max

source_name: Root

extract_protocol:
Hot Phenol RNA extraction from soybean root tissue
mRNA library preperation (polyA enrichment) - NoaSeq PE150 (12G raw data per sample)

tag: tissue

value: Root

value: Williams 82 soybean

tag: genotype

value: WT-GmMKK2 overexpression

value: Heterodera glycines


title: K1718-P-R1

value: parental


title: Sample 3_VL51-IDE-R-R1


title: WT-GmMKK2 H. glycines infected RNA bio rep 1 [W102R3_1]


title: Sample 1_VL51-P-R1

value: Parental


title: WT-3 (VCAM1 control)

city: Pittsburgh

first: Partha

last: Dutta

state: PA

address: 200 Lothrop Street

department: Medicine

institute: University of Pittsburgh

description: Spatial RNA seq using Slide Seq was performed

data_processing:
For the RNA seq analysis, CLC Genomics Workbench v 11.0.1 was used for RNA seq analysis. Partek Flow (version 10.0.22.1204) software was used to analyze the single cell RNA seq data using the count matrix generated after the sequencing of the libraries. The RNA-Seq analysis is done in several steps using CLC Genomics Workbench: First, all genes are extracted from the reference genome (using annotations of type gene). Other annotations on the gene sequences are preserved (e.g. CDS information about coding sequences etc). Next, all annotated transcripts (using annotations of type mRNA) are extracted. If there are several annotated splice variants, they are all extracted. Note that the mRNA annotation type is used for extracting the exon-exon boundaries. Next, the reads are mapped against all the transcripts plus the entire gene.
For the slide-seq data analysis, Seurat was employed for the quality control, normalization and clustering analyses. Cell type deconvolution of Slide-seq data was performed using RCTD. Single-cell RNA-seq reference data from vascular cells after 14 days of elastase exposure during abdominal aortic aneurysm were utilized.  Quality control, normalization, and clustering analyses were conducted using the Seurat package. Cell types were determined based on key markers identified in Zhao et al. To enhance deconvolution quality, the reference dataset was limited to the predominant cell types: macrophages (4 sub-clusters), fibroblasts (2 sub-clusters), and smooth muscle cells (2 sub-clusters). RCTD was executed in doublet mode, given the Curio Slide-seq's high resolution, with a Unique Molecular Identifier (UMI) threshold of 50 for default-aligned samples and 10 for short-read-aligned samples. Subsequent analyses included regions exclusively identified as macrophages with a weight ≥ 0.6 or as two cell types with a macrophage weight ≥ 0.6. For downstream differentially expressed gene (DEG) analysis, six Slide-seq datasets, formatted as Seurat objects, were integrated using the SCTransform pipeline of Seurat. The confidently labeled macrophage regions were extracted from the integrated Seurat object.  control samples within the macrophage population and its sub-clusters. Over-representation analysis (ORA) of DEGs was conducted using the WebGestalt online toolkit. DEGs with an adjusted p-value > 0.05 or an absolute log2 fold change < 1 were excluded. The murine canonical pathways database from MsigDB served as the functional database for the analysis.
From this mapping, the reads are categorized and assigned to the genes, and expression values for each gene and each transcript are calculated. After that, putative exons are identified.
Fold change in gene expression was calculated
Assembly: mm10
Supplementary files format and content: The excel file includes RPKM values for each Sample obtained from the RNA seq analysis. For the spatial transcriptomics data, we have included an RDS file containig the data for all macrophages.

organism: Mus musculus

source_name: Aortic root

extract_protocol:
We used pico pure RNA extraction kit (Applied Biosystems) to extract RNA from sorted cells. Additionally, we used Slide-seq (Applied Biotechnology Laboratory) to perform spatial trascriptomics.
RNA libraries were prepared for sequencing using standard Illumina protocols
RNA seq and spatial RNA seq

value: Aortic root

value: All cells

value: Ldlr-/- LysM+/+ Vcam1fl/fl

value: atherogenic diet


title: Sample 2_VL51-P-R2


title: WT-1 (VCAM1 control)


title: VCAM-1 KO-3

value: Ldlr-/- LysMcre/+ Vcam1fl/fl


title: VCAM-1 KO-2


title: IgG_CD31

city: Charlottesville

first: Gary

middle: K

last: Owens

state: VA

address: 415 Lane Road MR5 Building Room 1322

institute: University of Virginia

library_source: transcriptomic single cell

data_processing:
Basecalls performed using CASAVA
Reads were aligned against mouse genome mm10 using the CellRanger software (10x Genomics).
Gene-barcode matrices were analyzed in R using Seurat v3. Cells were filtered for 200-5000 reads per UMI, 10% or less mitochondrial and less than 5% hemoglobin gene content.
Significant principal components of variation (PCs) were calculated using JackStraw test with 10000 repetitions, and clusters were calculated as described in the manuscript
Differential expression analysis was done using MAST
Assembly: mm10
Supplementary files format and content: Cell-gene-barcode matrices

source_name: Atherosclerosis plaques

extract_protocol:
Individual atherosclerotic plaques from the brachiocephalic artery were removed from underlying media with forceps and deposited into FACS buffer (1% BSA in PBS) plus 1 ug/mL Actinomycin D,(Gibco, #11805017). Samples were then minced and digested with 1mL Liberase (Roche, #355374) plus 1ug/mL ActinomycinD for 1 hour at 37 degrees, then resuspended in PBS plus 0.04% UltraPure non-Acetylated BSA (ThermoFisher #AM2616) and filtered through 30um filters on ice. Samples were then either counted and submitted directly for scRNAseq or sorted by flow cytometry. Samples for flow were stained with Sytox Blue viability dye and sorted with a BD FACSAria Fusion cell sorter with gating on live, single cells that were eYFP+. Cells were collected into LoBind tubes containing 0.04% non-acetylated BSA.
2,000 cells in each group were targeted in Chromium 10X genomics libraries, which after barcoding, were pooled and sequenced on the Illumina NextSeqTM, 150 cycle high-output.
single-cell RNA-seq (10x genomics, 3')

treatment_protocol: Animals were maintened in a Western Diet for 18 weeks

value: Atherosclerosis plaques

value: CD31 Sorted cells

value: Apoe-/-


title: siControl-5

description: Atherosclerotic macrophages were sorted from Apoe KO mice after injecting them with siControl for one month

source_name: Aortic tissue

value: Aortic tissue

value: Macrophages


title: siControl-4


title: siControl-3


title: siControl-2


title: siCmpk2-5

description: Atherosclerotic macrophages were sorted from Apoe KO mice after injecting them with siCmpk2 for one month


title: siControl-1


title: siCmpk2-4


title: K1718-P-R2

value: B-cell


title: siCmpk2-2


title: siCmpk2-1


title: VCAM1 negative-6

description: VCAM-1 negative macrophages were sorted from atherosclerotic plaques


title: VCAM1 negative-2


title: VCAM1 negative-5


title: VCAM1 negative-1


title: VCAM1 negative-4


title: VCAM1 positive-5

description: VCAM-1 positive macrophages were sorted from atherosclerotic plaques


title: WT-2 (VCAM1 control)


title: VCAM1 positive-6


title: VCAM1 positive-2


title: VCAM1 negative-3


title: VCAM1 positive-3


title: VCAM1 positive-1


title: siCmpk2-3


title: SmoA1_Control_Sample2.4 biol rep 4

city: Toronto

first: Carolina

last: Nor

state: Ontario

address: 686 Bay St

department: Developmental & Stem Cell Biology

country: Canada

institute: The Hospital for Sick Children

data_processing:
Cell Ranger software v.5.0.1 default pipeline
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: brain tumor

extract_protocol:
RNA extraction was performed using a RNeasy Qiagen kit.
Ribodeplete protocol

value: brain tumor

tag: strain

value: SmoA1 homo

value: Sham

tag: time


title: SmoA1_Control_Sample1.4 biol rep 4


title: SmoA1_Control_Sample1.1 biol rep 1


title: SmoA1_Control_Sample4.1 biol rep 1


title: SmoA1_Control_Sample4.2 biol rep 2


title: SmoA1_Control_Sample2.1 biol rep 1


title: SmoA1_Control_Sample2.3 biol rep 3


title: SmoA1_Control_Sample4.4 biol rep 4


title: SmoA1_Control_Sample1.3 biol rep 3


title: SmoA1_Control_Sample4.3 biol rep 3


title: VCAM-1 KO-1


title: SmoA1_Control_Sample1.2 biol rep 2


title: SmoA1_1Gy_5hr_Sample3.3 biol rep 3


title: SmoA1_1Gy_5hr_Sample3.4 biol rep 4


title: SmoA1_1Gy_5hr_Sample3.2 biol rep 2


title: VCAM1 positive-4


title: SmoA1_1Gy_5hr_Sample2.4 biol rep 4


title: SmoA1_1Gy_5hr_Sample2.3 biol rep 3


title: SmoA1_1Gy_5hr_Sample1.4 biol rep 4


title: SmoA1_1Gy_5hr_Sample1.3 biol rep 3


title: SmoA1_1Gy_5hr_Sample2.2 biol rep 2


title: SmoA1_1Gy_5hr_Sample2.1 biol rep 1


title: SmoA1_1Gy_5hr_Sample1.2 biol rep 2


title: Ptc_Control_Sample_3 biol rep 3


title: SmoA1_1Gy_5hr_Sample1.1 biol rep 1


title: SmoA1_Control_Sample2.2 biol rep 2


title: Ptc_Control_Sample_2 biol rep 2


title: Ptc_Control_Sample_1 biol rep 1


title: Ptc_5h_Radiation_Sample_3 biol rep 3


title: BL6_Control_Sample1.4 biol rep 4

source_name: cerebellum

value: cerebellum


title: BL6_Control_Sample3.3 biol 3


title: BL6_Control_Sample3.2 biol 2


title: Ptc_5h_Radiation_Sample_2 biol rep 2


title: BL6_Control_Sample2.2 biol rep 2


title: BL6_Control_Sample2.3 biol rep 3


title: Ptc_5h_Radiation_Sample_1 biol rep 1


title: BL6_Control_Sample2.1 biol rep 1


title: BL6_Control_Sample3.4 biol rep 4


title: BL6_Control_Sample2.4 biol 4


title: BL6_Control_Sample1.1 biol rep 1


title: SmoA1_1Gy_5hr_Sample3.1 biol rep 1


title: BL6_Control_Sample1.2 biol rep 2


title: BL6_Control_Sample3.1 biol rep 1


title: BL6_1Gy_5hr_Sample4.4 biol rep 4


title: BL6_1Gy_5hr_Sample4.2 biol rep 2


title: BL6_1Gy_5hr_Sample3.4 biol rep 4


title: BL6_1Gy_5hr_Sample4.1 biol rep 1


title: BL6_1Gy_5hr_Sample3.3 biol rep 3


title: BL6_1Gy_5hr_Sample3.2 biol rep 2


title: BL6_1Gy_5hr_Sample3.1 biol rep 1


title: BL6_1Gy_5hr_Sample4.3 biol rep 3


title: BL6_1Gy_5hr_Sample2.2 biol rep 2


title: BL6_1Gy_5hr_Sample2.1 biol rep 1


title: BL6_1Gy_5hr_Sample2.3 biol rep 3


title: BL6_1Gy_5hr_Sample2.4 biol rep 4


title: 73577 (PLX5622 Rep. 4)

city: Jena

first: Philipp

last: Koch

address: Beutenbergstraße 11

department: Core Facility Life Science Computing

institute: Leibniz Institute on Aging - Fritz Lipmann Institute

data_processing:
Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422.
The RNA sequencing reads were aligned to the mouse reference genome (GRCm38 with the Ensembl genome annotation release 99) using STAR 2.7.2b (parameters: --alignIntronMax 100000, --outSJfilterReads Unique, --outSAMmultNmax 1, --outFilterMismatchNoverLmax 0.04) (PMID: 23104886)
For each gene, all reads that map uniquely to one genomic position were counted with FeatureCounts 1.6.5 (multi-mapping or multi-overlapping reads were discarded, stranded mode was set to “–s 2”) (PMID: 24227677)
Assembly: GRCm38
Supplementary files format and content: csv file containing raw read counts per gene over all samples

source_name: isolated brain CD11b cells

growth_protocol: Animals were housed under controlled/standardized day-night conditions (12h/12h) at room temperature (23±1°C, 30%–60% environmental humidity) and received a standard diet (AIN) or PLX5622 treatment and water ad libitum.

extract_protocol:
CD11b positive cells were isolated according to MACS cell separation protocol (Miltenyi Biotec). Total RNA extraction was carried out using QIAzol lysis reagent and RNeasy Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions.
Libraries were prepared from 300 ng of input material (total RNA)using NEBNext Ultra II Directional RNA Library Preparation Kit in combination with NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext® Multiplex Oligos for Illumina®(Index Primers Set 1/2/3/4) following the manufacturer's instructions (New England Biolabs).

treatment_protocol: Mice were treated with the colony-stimulating factor 1 recepto inhibitor PLX5622 for 7 days followed by 10 days of PLX5622 withdrawal in order to repopulate microglia

value: isolated brain CD11b cells

value: 17 days

tag: age

value: 10-13 weeks


title: 73542 (PLX5622 Rep. 3)


title: BL6_Control_Sample1.3 biol rep 3


title: 73501 (PLX5622 Rep. 2)


title: HIRE7

city: Richland

first: James

middle: Michael

last: Fulcher

state: Washington

address: 902 Battelle Blvd

department: Environmental Molecular Sciences Laboratory

institute: Pacific Northwest National Laboratory

data_processing:
Data quality was assessed with fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Read-trimming was conducted using bbduk (http://jgi.doe.gov/data-and-tools/bb-tools/).
Reads were aligned to the mouse genome using STAR (https://github.com/alexdobin/STAR).
BAM file outputs were mapped to genes using htseq‐count (Anders et al., 2015) using default settings.
Assembly: human genome GRCh38
Supplementary files format and content: .txt files represent number of raw read counts mapping to each gene

source_name: Human pancreatic islet cell

growth_protocol: Human pancreatic islets from two human donors (Prodo Laboratories) were acquired and cultured for 24 hr in pancreatic islet media (PIMS, Prodo Laboratories).

extract_protocol:
RNA was acquired through nanoSPLITS. RNA quality and quantity were analysed with a RNA6000 Nano Assay using the Agilent 2100 Bioanalyzer (Agilent Technologies).
For RNA‐Seq, The TruSeq stranded mRNA (cat#20020594) was used to generate cDNA library for illumina NextSeq550 platform according to the manufacture protocol. Single-read sequencing of the cDNA libraries with a read length of 150 was performed with NextSeq 500 Sequencing System using NextSeq 500/550 High Output v2 kit 150 cycles (cat#20024907).

treatment_protocol: Single cells were dissociated with TrypLE for ~15-30 minutes, washed with PBS, stained with Calcein-AM/propidium iodide, and sorted onto nanoSPLITS chips using a cellenONE cell sorter.

value: Human pancreatic islet cell

value: primary cells from 2 human donors

tag: medium

tag: time point

value: 24 hour culture


title: HIRE4

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: HIRE5


title: HIRE9


title: 73401 (PLX5622 Rep. 1)


title: HIRE11


title: HIRE8


title: HIRE12


title: HIRE1


title: HIRE10


title: HIRE6


title: HIRE2


title: HIRE3


title: HIRF12


title: HIRF8


title: HIRF10


title: HIRF9


title: HIRF6


title: HIRF7


title: HIRF4


title: HIRF5


title: HIRF1


title: HIRG12


title: HIRF2


title: HIRF3


title: HIRG11


title: HIRG10


title: HIRG9


title: HIRF11


title: HIRG5


title: HIRG6


title: HIRG4


title: HIRG7


title: HIRG2


title: HIRG3


title: HIRH7


title: HIRH12


title: HIRH8


title: HIRG1


title: HIRH9


title: HIRH10


title: HIRH6


title: HIRH5


title: HIRH3


title: HIRH1


title: HIRG8


title: HIRH2


title: HIRA10


title: HIRA12


title: HIRA9


title: HIRA11


title: HIRA8


title: HIRA6


title: HIRA5


title: HIRA4


title: HIRA3


title: HIRA1


title: HIRA2


title: HIRH11


title: HIRB10


title: HIRB12


title: HIRB11


title: HIRB9


title: HIRH4


title: HIRB4


title: HIRA7


title: HIRB1


title: HIRB8


title: HIRB3


title: HIRB2


title: HIRB6


title: HIRB7


title: HIRC7


title: HIRC9


title: HIRC10


title: HIRC5


title: HIRC6


title: HIRC11


title: HIRC4


title: HIRC3


title: HIRC2


title: HIRC1


title: HIRD11


title: HIRD10


title: HIRD9


title: HIRD8


title: HIRD12


title: HIRD6


title: HIRD7


title: HIRD5


title: HIRD4


title: HIRC12


title: HIRD3


title: HIRC8


title: HIRD2


title: HIRD1


title: HIRB5


title: Chip1D11


title: Chip1D12


title: Chip1D2


title: Chip1D7


title: Chip1D4


title: Chip1D1


title: Chip1D5


title: Chip1D9


title: Chip1C12


title: Chip1C11


title: Chip1D3


title: Chip1C10


title: Chip1C9


title: Chip1C8


title: Chip1C7


title: csr1_RNAi_rep2

city: Saskatoon

first: Cheng-Wei

last: Wu

state: SK

address: 52 Campus Drive

institute: University of Saskatchewan

description:
csr1_2
Wu 2023 csr-1 processed data.xls

data_processing:
HISAT2 v2.0.5 used for mapping to the C. elegans reference genome
FeatureCounts v1.5.0-p3 used for Quantification
rMATS v3.2.5 used for Alternative Splicing analysis
StringTie v1.3.3 used for novel gene prediction.
DESeq2 v1.20.0 used for differential expression analysis
Assembly: wbcel235
Supplementary files format and content: Excel file includes differential gene expression and alternative splicing for each file

organism: Caenorhabditis elegans

source_name: whole animal

extract_protocol:
Total RNA extracted using the RNAqueous Total RNA Isolation Kit
Standard Illumina protocols with oligo-dT beads

value: whole animal

value: csr-1 RNAi


title: csr1_RNAi_rep3

description:
csr1_3
Wu 2023 csr-1 processed data.xls


title: Chip1D10


title: Chip1D6


title: Chip1D8


title: Chip1C4


title: EV_RNAi_rep2

description:
EV_2
Wu 2023 csr-1 processed data.xls

value: empty vector


title: Chip1C6


title: csr1_RNAi_rep1

description:
csr1_1
Wu 2023 csr-1 processed data.xls


title: EV_RNAi_rep1

description:
EV_1
Wu 2023 csr-1 processed data.xls


title: Chip1C5


title: Chip1C3


title: Intestine, uninfected control of WT H18N11 experiment, bat 2

city: Fort Collins

first: Tony

last: Schountz

address: 3185 Rampart Rd

institute: Colorado State University

data_processing:
A custom reference genome, appending H18N11 sequences to the Artibeus jamaicensis genome (Cold Spring Harbor Laboratories genome, NCBI RefSeq assembly GCF_021234435.1), was built with cellranger (v 6.1.2) using mkref function.
cellRanger count function was used to process the fastq files using default parameter settings.
Assembly: CSHL_Jam_final
Supplementary files format and content: raw h5 file including feature count per sample

organism: Artibeus jamaicensis

source_name: Intestine

extract_protocol:
The small and large intestines were cut longitudinally to expose the lumen, intestines were then cut into 0.5 to 1.0 cm pieces and washed four times with 10 mL wash solution consisting of 1×PBS, 5 mM EDTA under vigorous shaking. Subsequently, 20mL of predigestion buffer (Hanks' balanced salt solution (HBSS) containing 20mM HEPES, 5mM EDTA, 5% fetal calf serum (FCS) and 1mM Dithiothreitol (DTT)) were added and incubated at 37°C for 20minutes with constant rotation. After vortexing for 10 sec, the pieces were drained through a 70-µm cell strainer and the supernatant was saved. Cells in the supernatant were washed three times by adding wash solution and subsequent centrifugation at 350g for 5min. The predigestion step was then repeated as described above. After the second round of predigestion, 20mL of HBSS containing 20mM HEPES was added to the intestinal pieces and incubated for 20 min under continuous rotation followed by vigorous vortexing for 10sec. The pieces were sieved through a 70-µm cell strainer, and the supernatant was washed with wash solution as described above. All cell fractions contained epithelial cells and were pooled. Intestinal pieces were resuspended in digestion buffer (HBSS with 20mM HEPES, 1.25mM CaCl2, 1mM MgCl2, 5mM EDTA, 5% FCS, Lamina Propria Dissociation Kit enzymes D, R, and A, were added according to the manufacture’s protocol) and incubated at 37°C for 30 min with constant rotation. The intestinal tissue was transferred into a gentleMACS C Tube and processed with a gentleMACS using the manufacturer’s program. Perfusion solution was added and the pieces were strained through a 70-µm cell strainer. The supernatant was processed and washed as described above. This second fraction contained the cells of the lamina propria. Intraepithelial and lamina propria fractions of each bat were counted using trypan blue and cells were mixed in a 1:1 ratio for each bat. To prepare a single-cell suspension of the mesentery, the mesentery was tamped through a cell strainer in a Petri dish containing 5 mL of perfusion solution.
A total of 12,000 cells per sample were processed with a Chromium Next GEM Single Cell 5' Kit v2 (10x Genomics) to achieve a recovery of at least 8000 cells.

value: Intestine

tag: breed

value: outbreed

tag: Sex

value: male

tag: health condition

value: healthy

value: uninfected control


title: Chip1C1


title: MLKL3

city: Fuzhou

first: Chengyu

last: Liao

state: Fujain

address: 134, east road

department: Shengli Clinical Medical College

country: China

institute: Fujian Medical University

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files

source_name: pancreatic orthotopic tumor cells

growth_protocol: KPC cells with Vector or MLKL overexpression were injected to construct C57bl/6 pancreatic orthotpic model then harvested for scRNA-seq. Mice were observed the liver metastasis through micro-CT and Small Animal In Vivo Imaging.

extract_protocol:
freshly collected pancreatic orthotopic tumors from C57BL/6 mice injected with KPC-OE-Vector cell or KPC-OE-MLKL cells were flushed with ice-cold PBS  then performed by  digestor. The RBC cells were removed and the isolated cells were resuspended in digestion buffer (HBSS with Mg2+/Ca2+ supplemented with 2mg/ml collagenase type I and 40 U/ml DNase I) and incubated at 37℃ for 30min. Cells were washed and re-suspended in PBS and stained with a vialility dye to evalutaed the cell viability.
library was performed according to the manufacter's instructions (single cell 3'v2 protocol, 10x Genomics). Briefly, cells were respended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion(GEM). the poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA. the pooled barcoded cDNA was then cleaned up with silane dynabeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction.

value: pancreatic orthotopic tumor cells

value: KPC-OE-MLKL

value: pancreatic cancer cells


title: Chip1C2


title: MLKL2

description: 10x Genomics

treatment_protocol: KPC cells with Vector or MLKL overexpression were injected to construct C57bl/6 pancreatic orthotpic model then harvested for scRNA-seq.


title: Chip1B12


title: HK2_ROSU_2

type: RNA

city: Innsbruck

first: Johannes

last: Leierer

address: Anichstr. 35

department: Internal Medicine IV - Nephrology

country: Austria

institute: Medical University Innsbruck

hyb_protocol: Hybridization to oligonucleotide microarrays (Human GE 4x44K v2, G4845A) according to manufacturers protocol

scan_protocol: Arrays were scanned at 5 µm using an Axon Gene Pix 4000B scanner (Molecular Devices, Sunnyvale, CA) and signal intensity data extracted using Agilent Feature Extraction software (v.9.5.3.1).

data_processing: Data preprocessing consisted of (i) excluding low abundance features (intensity values < 1.5 above background signals), (ii) quantil-quantil data normalization, and (iii) summarization of identical sequence spots.

label_protocol: Cyanine 3 (Cy3) labelled cRNA was generated with the LowInput QuickAmp Labeling Kit (5190-2305) for hybridization to oligonucleotide microarrays (Human GE 4x44K v2, G4845A).

growth_protocol: Proximal tubular human kidney cells (HK2) were cultured in Keratinocyte-Serum Free Medium (KSFM) containing 10% fetal bovine serum (FBS), 5 ng/ml recombinant epidermal growth factor (rEGF), 0.05 mg/ml bovine pituitary extract (BPE), 100 U/ml penicillin and 100µg/ml streptomycin. Mesangial cells (MC) were cultured in MsBM Mesangial Basal Medium containing 5% FCS, 30 µg/ml and 15 ng/ml Amphotericin B. All cells were grown at 37 °C in a humidified atmosphere with 5% CO2.

extract_protocol: After 24 hours total RNA was isolated with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol

treatment_protocol: After growth to confluency, cells were serum deprived for 24 hours and stimulated with 30mM glucose for 24 hours. Thereafter cells were either treated with 20 µM atorvastatin or rosuvastatin for 24 hours or left untreated.

value: Rosuvastatin

value: proximal tubule cell line


title: HK2_ROSU_1


title: HK2_ATO_3

value: Atorvastatin


title: MLKL1

description: 10x Genomics


title: Intestine, uninfected control of delNS1 experiment, bat 2


title: EV_RNAi_rep3

description:
EV_3
Wu 2023 csr-1 processed data.xls


title: HK2_CTRL_3

value: control


title: HK2_ATO_1


title: HK2_CTRL_1


title: HK2_ROSU_3


title: HK2_CTRL_2


title: Chip1B11

value: Human pancreatic islet cell

value: primary cells from 2 human donors

value: 24 hour culture


title: HK2_ATO_2


title: Vec3

description: 10x Genomics

value: KPC-OE-Vector


title: Intestine, uninfected control of delNS1 experiment, bat 1


title: Chip1B10

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: Vec2

description: 10x Genomics


title: Chip1B9


title: MC_ROSU_3

source_name: Mesangial cells

value: mesangial cell line

value: MC


title: Vec1

description: 10x Genomics


title: Chip1B8


title: Intestine, 9 dpi with delNS1, bat 2

value: Intestine

value: outbreed

value: male

value: infected with delNS1


title: Intestine, uninfected control of WT H18N11 experiment, bat 1

value: healthy

value: uninfected control


title: MC_ROSU_2

value: Rosuvastatin


title: SSDS-RPA2_Nbs1dB_rep2

city: New York

first: Soonjoung

last: Kim

email: kims16@mskcc.org, soonjk@yuhs.ac

state: New York

address: 430 East 67th Street

department: Molecular Biology

institute: Memorial Sloan Kettering Cancer C

library_source: genomic

data_processing:
We then ran a previously described bioinformatic pipeline on the resulting reads for identification of single-stranded sequences (ref. 1). Sequence reads at SPO11-dependent hotspots were analyzed using R version 4.0.3.  <http://www.r-project.org>.  (ref. 1) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018).
Sequence reads were mapped onto the mouse reference genome (mm10).
Reads for ssDNA type 1 of ChIP-seq that were uniquely and properly mapped were counted. ssDNA type 1 has both 5′ and 3′ overhangs when hairpin structure is formed, the end-repair enzyme mix of Klenow and T4 polymerases will fill in the protruding 5′ end after the exonucleolytic digestion of the 3′ overhang that gives unique inverted repeats at the ends (ref. 1).Maps were further analyzed using R versions 4.0.3. <http://www.r-project.org>.  (ref. 1) Khil, et al., Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA, Genome Res. May; 22(5): 957–965. (2012).
Assembly: mm10
Supplementary files format and content: Bed files contain mapped tag densities across the mappable portion of the genome, specific for ssDNA type 1. Reads were not normalized to RPM (reads per million mapped).

molecule: genomic DNA

source_name: testis

extract_protocol:
ChIP SSDS was performed following the previously published (ref. 1, 2, 3). Fresh or frozen testes of wild-type or Nbs1∆B were placed in room temperature 1% PFA (16% stock, Pierce) in PBS and incubated for 15 min with gentle agitation. The crosslinking was stopped by adding 2.5 M glycine to a final concentration of 125 mM, followed by 5 min incubation at room temperature. Fixed cells were immediately homogenized in a Dounce homogenizer with 20 strokes of the tight-fitting pestle, then passed through a 70 μm cell strainer. The nuclei were prepared as described (ref. 2), resuspended in 900 μl shearing buffer (0.1% SDS, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) supplemented with protease inhibitor (Roche), split into two ∼500 μl aliquots and sonicated to fragment sizes ranging 500–1000 bp with a Covaris system (E220 Focused-ultrasonicator, microtube-500) using the following parameters: delay 300 s then three cycles of [peak power 75 W, duty factor 10%, cycles/burst 200, duration 150 s, and delay 60 s]. Sheared nuclei were centrifuged at 12,000 g for 10 min at 4 °C to remove debris. The sonicated chromatin was then transferred to 3 ml Slide-A-Lyzer G2 dialysis cassette (Pierce) and dialyzed with ChIP buffer (16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) for 5 hr at 4 °C with constant but slow stirring. For each immunoprecipitation, 20 μl of anti-DMC1 (Abcam, ab11054), 10 μl of anti-RAD51 (Novus Biologicals, NB100-148), or 10 μl of anti-RPA2 (Abcam, ab76420) was added to chromatin lysates and incubated overnight at 4 °C with end-over-end rotation.  As described previously (ref. 1, 2, 3), protein G or A Dynabeads were added to chromatin and antibody mixture and incubated 2 hr at 4 °C, then immunoprecipitated DNA was washed and eluted.  (ref. 1) Khil, et al., Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA, Genome Res. May; 22(5): 957–965. (2012).  (ref. 2) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018).  (ref. 3) Hinch, et al., The Configuration of RPA, RAD51, and DMC1 Binding in Meiosis Reveals the Nature of Critical Recombination Intermediates. Mol Cell 79(4): 689-701 e610. (2020)
To reverse DNA-protein crosslinks, 12 μl of 5 M NaCl was added to 300 μl of eluted sample and incubated overnight at 65 °C. DNA was cleaned with MinElute PCR Cleanup Kit (Qiagen) after proteinase K (Thermo) treatment. Following end repair and dA-tailing, DNA was incubated at 95 °C for 3 min and cooled to room temperature to enrich for single-stranded DNA as described before (ref. 1). After ligation with sequencing adaptors, SSDS library was amplified with 12 cycles of PCR.   After Qubit quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina sequencer. We obtained paired-end reads of 100 bp. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes. Base calls were perfomed using bcl2fastq software v.2.20 or bcl-convert software v.3.9.3 to 3.10.5 for samples sequenced on Illumina NovaSeq 6000.  (ref. 1) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018).

value: testis

value: Spermatocytes

tag: chip antibody


title: Intestine, 9 dpi with delNS1, bat 1


title: Chip1B7

tag: cell type


title: SSDS-RPA2_Nbs1dB_rep1


title: Chip1B6


title: Intestine, 3 dpi with delNS1, bat 3

tag: treatment


title: MC_ATO_3

value: Atorvastatin


title: Intestine, 3 dpi with delNS1, bat 2


title: SSDS-RAD51_Nbs1dB_rep3


title: MC_ATO_2


title: SSDS-RAD51_Nbs1dB_rep2


title: Intestine, 3 dpi with delNS1, bat 1


title: Chip1B5

value: Human pancreatic islet cell


title: Intestine, 9 dpi with delNS1, bat 3


title: SSDS-RAD51_Nbs1dB_rep1


title: SSDS-DMC1_Nbs1dB_rep2


title: Artificial sequences, 1M7 treated sample, 5mM Mg, 1mM SAM, replicate 0

city: Paris

first: Pierre

last: HARDOUIN

state: France

address: 4 avenue de l'observatoire

department: CiTCoM

country: France

institute: Faculté de pharmacie UMR 8038

description: dsDNA library, 500 SAM-I aptamer artificial sequences

library_source: other

data_processing:
The demultiplexing is made using cutadapt. (https://cutadapt.readthedocs.io/en/stable/)
Quality trimming, Paired read merging, Sequence alignment, Mutation identification, Mutation counting, Sequence correction, reactivity profile calculation were made by using Shapemapper 2 (https://github.com/Weeks-UNC/shapemapper2)
Assembly: N/A
Supplementary files format and content: Tab-separated values files

molecule: other

organism: synthetic construct

source_name: synthetic construct

extract_protocol:
N/A
RNAs were in vitro transcribed, modified with SHAPE reagent and reverse transcribed with the Superscript III reverse transcriptase (Invitrogen®) and NGS libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (New England Biolabs®) as manufacter’s instructions.

value: synthetic construct


title: Chip1B4

value: primary cells from 2 human donors

value: 24 hour culture


title: Intestine, 9 dpi with WT H18N11, bat 3

value: infected with WT H18N11


title: Chip1B2

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: Chip1B3


title: MC_ATO_1

value: mesangial cell line

value: MC


title: Intestine, 9 dpi with WT H18N11, bat 2

value: Intestine

value: outbreed


title: Artificial sequences, 1M7 treated sample, 5mM Mg, 0,1mM SAM, replicate 0


title: Intestine, 9 dpi with WT H18N11, bat 1

value: male


title: Chip1B1


title: SSDS-RPA2_wt_rep2

value: wild type


title: Chip1A12


title: Artificial sequences, 1M7 treated sample, without Mg, without SAM, replicate 0


title: MC_CTRL_3

value: control


title: Chip1A11


title: SSDS-RAD51_wt_rep2


title: Intestine, 3 dpi with WT H18N11, bat 2

value: healthy


title: MC_ROSU_1

value: Rosuvastatin


title: MC_CTRL_2


title: Artificial sequences, 1M7 treated sample, 5mM Mg, without SAM, replicate 0


title: Chip1A10


title: Intestine, 3 dpi with WT H18N11, bat 1


title: SSDS-RAD51_wt_rep1

value: testis

value: Spermatocytes


title: Artificial sequences, Untreated sample, DMSO, replicate 0


title: SSDS-DMC1_Nbs1dB_rep1


title: Chip1A9

value: Human pancreatic islet cell


title: Mesentery, uninfected control of delNS1 experiment, bat 2

source_name: Mesentery

value: Mesentery

value: uninfected control


title: SSDS-DMC1_wt


title: Chip1A8

value: primary cells from 2 human donors

value: 24 hour culture


title: Mesentery, uninfected control of delNS1 experiment, bat 1


title: ENL_Y78A

city: Tianjin

first: 爱源

last: 王

state: —Please choose an option—

address: No. 22, Meteorological Observatory Road, Heping District, Tianjin

department: Department of Biochemistry and Molecular Biology

institute: TianjinMedical University

data_processing:
Raw reads were mapped to the human reference genome (hg19) and transcriptome using the RNA-Seq unified mapper. Read counts for each transcript were calculated using HTseq v0.6.1 using default parameters54. Differential gene expression analyses were performed using the “exactTest” function in edgeR v3.055
Assembly: hg19
Supplementary files format and content: rawcounts ; FPKM

extract_protocol:
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA).
Briefly, mRNA was purified from total RNA using poly-T  oligo-attached magnetic beads. Fragmentation was carried out using  divalent cations under elevated temperature in First Strand Synthesis  Reaction Buffer(5X). First strand cDNA was synthesized using random  hexamer primer and M-MuLV ReverseTranscriptase(RNase H-).Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were  ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were  purified with AMPure XP system. Then PCR was performed with  Phusion High-Fidelity DNA polymerase, Universal PCR primers and  Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer  2100 system.


title: ENL_wt


title: S1-seq_wt_timecourse_16dpp__rep1

data_processing:
After Qubit quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina sequencer. We obtained paired-end reads of 50 bp. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes. Base calls were perfomed using bcl2fastq software v.2.20 or bcl-convert software v.3.9.3 to 3.10.5 for samples sequenced on Illumina NextSeq 500, NextSeq 2000, or HiSeq 4000.  Reads were trimmed and filtered by Trim Galore version 0.6.6 with the arguments --paired --length 15 <http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/>. Sequence reads were mapped onto the mouse reference genome (mm10) by bowtie2 version 2.3.5.1 (ref. 1) with the arguments -N 1 -X 1000.  (ref. 1) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).
Duplicated reads were removed by Picard <https://broadinstitute.github.io/picard/>. Uniquely and properly mapped reads (MAPQ ≥ 20) were extracted by samtools with the arguments -q 20 <http://www.htslib.org/>.
Reads for S1, Exo7/T, or ExoT-seq were counted at which a nucleotide next to biotinylated adaptor DNA was mapped (this corresponds to a position of ssDNA). Maps were analyzed using R version 4.0.3 <http://www.r-project.org>.
Assembly: mm10
Supplementary files format and content: BigWig files contain read counts at positions of resection endpoints. Reads were not normalized to RPM (reads per million mapped). Letter "F" or "R" in sample names indicates that reads were mapped on forward or reverse strand, respectively.

extract_protocol:
Testis cells from mice at indicated age were obtained as described previously (ref. 1). Cells were embedded in plugs of low-melting point agarose to protect DNA from shearing, and DNA was liberated by treatment with SDS and proteinase K as described previously (ref. 1).  (ref. 1) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020).
In-plug overhang removal with S1 endonuclease for S1-seq, exonuclease VII and exonuclease T for Exo7/T-seq, or exonuclease T for ExoT-seq, and adaptor ligation were performed as described previously (ref. 1, 2, 3) with minor modifications. In brief for S1-seq, plugs were washed in 0.5 ml of modified S1 buffer (50 mM sodium acetate, 280 mM NaCl, 4.5 mM ZnSO4, pH 4.7) for 30 min four times, treated with 9 units of S1 nulcease in 500 µl of modified S1 buffer at 37°C for 20 min and washed once with 1ml TE. For Exo7/T-seq, plugs were washed in 1 ml of exonuclease VII buffer for 15 min twice, treated with 50 units of exonuclease VII in 100 µl of exonuclease VII buffer at 37°C for 60 min and washed once with 1ml TE and followed by exonulcease T treamtment. For ExoT-seq, plugs were washed in 1 ml of NEBuffer 4 for 15 min 3 times, treated with 75 units of exonuclease T in 100 µl of NEBuffer 4 at 24°C for 90 min and washed 3 times with 8 ml of TE for 15 min. The nuclease treated ssDNA ends were further polished with T4 polymerase before the first adaptor ligation as previously described (ref. 1, 2, 3). After ligation to biotinylated adaptors, DNA was purified from the agarose, sheared by sonication, purified with streptavidin, ligated to second-end adaptors, amplified, and sequenced as described previously (ref. 1, 2, 3).  (ref. 1) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020).  (ref. 2) Mimitou et al., S. A global view of meiotic double-strand break end resection. Science 355, 40-45 (2017).  (ref. 3) Mimitou et al., S1-seq Assay for Mapping Processed DNA Ends. Methods Enzymol 601, 309-330 (2018).
S1-seq, Exo7/T-seq, ExoT-seq

value: 16 days post partum


title: Intestine, 3 dpi with WT H18N11, bat 3

value: infected with WT H18N11


title: Mesentery, 9 dpi with delNS1, bat 3

value: infected with delNS1


title: MC_CTRL_1

value: mesangial cell line

value: MC


title: MZ_Cotwin2_clone2

city: La Jolla

first: Jingting

last: Yu

state: CA

address: 10010 N Torrey Pines Rd

institute: Salk Institute for Biological Studies

data_processing:
TherawfastqreadsunderwentsequencealignmentusingtheSplicedTranscriptsAlignmenttoaReference (STAR) and HOMER.Both raw count and transcripts per million (tpm) quantified matrix for all experiments were generated.
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw count for each sample
Supplementary files format and content: tab-delimited text file includes TPM values for each sample

molecule: polyA RNA

source_name: iPSC

extract_protocol:
We have prepared the neurons for RNA-seq according to apublished FACS protocol.Briefly, five- week-old neurons weredissociated and CD184−/CD44−/CD15-/CD24+cells (Miltenyi Biotec,cat. number 130-103-868, 130-113-334, 560828, 130-099-399, respectively) were collected and subjected to StrandedmRNA (PolyA+)-SeqLibraryPrep.
Atotalof1000ng ofRNAwasusedforlibrarypreparation using the Illumina TruSeq RNA Sample Preparation Kit. The libraries weresequenced on Illumina HiSeq2500 with 50 bp single-end reads.

value: iPSC


title: Chip1A7

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: S1-seq_wt_timecourse_15dpp__rep1

value: wild type

value: 15 days post partum


title: Artificial sequences, 1M7 denaturated sample, without Mg, without SAM, replicate 0

value: synthetic construct


title: SSDS-RPA2_wt_rep1

value: testis


title: Mesentery, 9 dpi with delNS1, bat 1

value: outbreed

value: male

value: healthy


title: MZ_Cotwin2_clone1


title: S1-seq_wt_timecourse_14dpp__rep1

value: Spermatocytes

value: 14 days post partum


title: Chip1A5

value: Human pancreatic islet cell

value: primary cells from 2 human donors

value: 24 hour culture


title: MZ_Cotwin1_clone2


title: Chip1A4


title: S1-seq_wt_timecourse_13dpp__rep1

value: 13 days post partum


title: S1-seq_wt_timecourse_12dpp__rep1

value: 12 days post partum


title: MZ_Cotwin1_clone1


title: Chip1A3


title: Mesentery, uninfected control of WT H18N11 experiment, bat 2

value: Mesentery

value: uninfected control


title: Lung, JJ011

first: Jian

last: Ge

institute: Columbia University

data_processing:
10x genomics' cellranger software version 6.1.2 was used to process the data.
there are few cell types other than lymphatic endothelial cells, need filter before cluster.
Assembly: mm10-2020-A
Supplementary files format and content: filtered_feature_bc_matrix.h5

source_name: lung

value: lung

value: lymphatic endothelial cells

value: Prox1-CreERT2; R26RtdT

tag: infection

value: 10 days post of influenza infection


title: Chip1A2

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: Mesentery, 9 dpi with WT H18N11, bat 3

value: outbreed

value: infected with WT H18N11


title: Lung, JJ010

value: non viral infection


title: MZ_Patient2_clone2

value: iPSC


title: MRE11-ChIP_AtmKO

data_processing:
After Qubit quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina sequencer. We obtained paired-end reads of 100 bp. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes. Base calls were perfomed using bcl2fastq software v.2.20 or bcl-convert software v.3.9.3 to 3.10.5 for samples sequenced on Illumina NovaSeq 6000.  Reads were trimmed and filtered by Trim Galore version 0.6.6 with the arguments --paired --length 15 <http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/>
Sequence reads were mapped onto the mouse reference genome (mm10) by bowtie2 version 2.3.5.1 (ref. 1) with the arguments -N 1 -X 1000.  Duplicated reads were removed by Picard <https://broadinstitute.github.io/picard/>. Uniquely and properly mapped reads (MAPQ ≥ 20) were extracted by samtools with the arguments -q 20 <http://www.htslib.org/>. (ref. 1) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).
Reads for MRE11 ChIP-seq that were uniquely and properly mapped were counted. Sequence reads at SPO11-dependent hotspots were analyzed using R version 4.0.3.  <http://www.r-project.org>.
Assembly: mm10
Supplementary files format and content: BigWig files contain mapped tag densities across the mappable portion of the genome. Reads were not normalized to RPM (reads per million mapped).

extract_protocol:
MRE11-ChIP was performed following the previously published DISCOVER-seq protocol (ref. 1) Fresh testes of 7 weeks old wild-type, Atm–/–, or Mre11-cHN mice were dissociated into single cells as described before (ref. 2) and resuspended in DMEM. Testis cells from mice at indicated age and genotype were obtained as described previously (ref. 1). PFA (16% stock, Pierce) was added to a final concentration of 1% and incubated for 15 min with gentle agitation, then the crosslinking was stopped by adding 2.5 M glycine to a final concentration of 125 mM, followed by 3 min incubation on ice. The nuclei were prepared as described (ref. 1) and up to 10 million nuclei were resuspended in 500 μl lysis buffer (0.5% N-lauroylsarcosine, 0.1% sodium deoxycholate, 10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA) supplemented with protease inhibitor (Roche), and sonicated to fragment sizes ranging 200–300 bp with a Covaris system (E220 Focused-ultrasonicator, microtube-500) using the following parameters: delay 300 s then six cycles of [peak power 75 W, duty factor 10%, cycles/burst 200, duration 150 s, and delay 60 s]. Sheared nuclei were centrifuged at 20,000 g for 10 min at 4 °C to remove debris and Triton-X (1% (vol/vol) final concentration) and lysis buffer were added to make it a total volume of 3 ml. (ref. 1) Wienert, et al., CRISPR off-target detection with DISCOVER-seq, Nat Protoc 15(5): 1775-1799 (2020). (ref. 2) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020).
For each immunoprecipitation, 10 μl of anti-MRE11 (Novus Biologicals NB100-142) was pre-mixed with 100 μl protein A Dynabeads, then added to chromatin lysates and incubated overnight at 4 °C with end-over-end rotation. Immunoprecipitated DNA was eluted and crosslinks were reversed as described (ref. 1). ChIP sequencing libraries were prepared from cleaned DNA as described for SSDS libraries (ref. 2) without the boiling step after the end repair and dA-tailing steps. After ligation with sequencing adaptors, the library was amplified by 15 cycles of PCR. The amplified library was purified.  Reads for S1, Exo7/T, or ExoT-seq were counted at which a nucleotide next to biotinylated adaptor DNA was mapped (this corresponds to a position of ssDNA). Maps were analyzed using R version 4.0.3 <http://www.r-project.org>.   (ref. 1) Wienert, et al., CRISPR off-target detection with DISCOVER-seq, Nat Protoc 15(5): 1775-1799 (2020). (ref. 2) Brick, et al., Analysis of Meiotic Double-Strand Break Initiation in Mammals. Methods Enzymol 601: 391-418. (2018). (ref. 3) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).

value: testis

value: Spermatocytes

value: Atm knockout

value: 7 weeks old


title: MZ_Patient1_clone2


title: Chip1A1

value: Human pancreatic islet cell

value: primary cells from 2 human donors

value: 24 hour culture


title: Mesentery, 9 dpi with WT H18N11, bat 1

value: Mesentery

value: male

value: healthy


title: MRE11-ChIP_Mre11cHN_rep3


title: MZ_Patient1_clone1


title: Chip2D12


title: Chip2D11


title: Chip1A6


title: Mesentery, 3 dpi with delNS1, bat 3

value: infected with delNS1


title: MRE11-ChIP_Mre11cHN_rep2


title: Mesentery, 3 dpi with delNS1, bat 1


title: Control3_2


title: Sequencing Lane 2, V(D)J

first: Jahan

last: Rahman

address: 417 E 68th St

department: Center for Hematologic Malignancies

institute: Memorial Sloan Kettering

description: SV-1840_SV_LN2_cell_sample_assign.csv.gz

data_processing:
Single cell RNA data were processed using Cellranger v6.0.2 count workflow to generate gene expression count matrices. Feature barcoding libraries were used to generate ADT count matrices and demultiplex cells/ perform cell-to-sample assignment with Cite-seq-count v1.4.5.  TCR data was processed using Cellranger’s vdj pipeline to generate cell-clonotype annotations.
Assembly: Human reference, GRCh38 (GENCODE v32/Ensembl 98)
Supplementary files format and content: Raw count matrices:  RNA_counts.csv.gz, ADT_counts.csv.gz
Supplementary files format and content: Filtered contig annotations: filtered_contig_annotations.csv.gz
Supplementary files format and content: Cell-sample annotations: cell_sample_assign.csv.gz
Library strategy: CITE-seq

source_name: Peripheral Blood Mononuclear Cells

extract_protocol:
PBMCs were thawed, stained with a viability dye (GhostDye 510) and an antibody targeting CD45, and sorted for viable (GhostDye-negative), CD45+ cells.
Standard library construction was performed using a 10x Chromium Single Cell Immune Profiling Gel Bead Kit.
Single-cell immune profiling (RNA-Seq, surface proteomics, and TCR-seq)

value: Peripheral Blood Mononuclear Cells

tag: library type

value: TCR-V(D)J

tag: antibodies/tags


title: MZ_Patient2_clone1


title: Mesentery, 3 dpi with WT H18N11, bat 1

value: outbreed


title: Sequencing Lane 2, Feature-barcoding

molecule: protein

value: ADT/HTO

value: Total-Seq C ADT Cocktail  and Total-Seq C hashing oligos


title: Chip2D9

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: MRE11-ChIP_Mre11cHN_rep1

value: testis


title: Mesentery, 9 dpi with WT H18N11, bat 2

value: infected with WT H18N11


title: MRE11-ChIP_wt_rep2

value: Spermatocytes

value: wild type


title: Control2_1

value: iPSC


title: Chip2D7

value: Human pancreatic islet cell

value: primary cells from 2 human donors


title: MRE11-ChIP_wt_rep1

value: 7 weeks old


title: Control3_1


title: Control1_2


title: Sequencing Lane 1, V(D)J

description: SV-1840_SV_LN1_cell_sample_assign.csv.gz


title: Mesentery, 3 dpi with WT H18N11, bat 3

value: Mesentery

value: male


title: Control2_2


title: SmoA1_braintumor_Sham_repB2

description: 10x Genomics

extract_protocol:
Brain tumors were dissected from mice, dissociated into single cell suspensions using papain and were run on the 10X Genomics Chromium Controller using the Chromium Single Cell 3' Solution V3 and V3.1 protocols
10x Genomics Chromium Single Cell 3' Solution V3 and V3.1 protocols

value: brain tumor

tag: mouse line

value: SmoA1 homo

value: Sham


title: Sequencing Lane 1, Feature-barcoding

value: Peripheral Blood Mononuclear Cells


title: Chip2D6

value: 24 hour culture


title: SmoA1_braintumor_Sham_repA5

description: 10x Genomics

tag: tissue


title: Chip2D10

tag: genotype


title: Chip2D5


title: Sequencing Lane 1, RNA

value: mRNA


title: SmoA1_braintumor_Sham_repA1

description: 10x Genomics


title: Chip2D4

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: SmoA1_braintumor_IR_5h_repC2

description: 10x Genomics


title: Chip2D8


title: Chip2D3


title: H3K9cr

data_processing:
FastQC tool was used to examine metrics, such as sequencing error rate, sequencing depth, and fragment length distribution
Aligned the preprocessed reads to a reference genome using alignment tool (Bowtie2)
Peak calling was performed using the model-based analysis of ChIP–seq (MACS2)
BigWig files were generated through Deeptools
Assembly: hg38
Supplementary files format and content: bigWig narrowPeak
Library strategy: CUT&Tag

extract_protocol:
HepG2 cells  were collected and counted for incubation with pre-treated ConA beads. Subsequently, cells are resuspended in an Antibody Buffer and incubated overnight at 4 °C with the corresponding primary antibody . The secondary antibodies were diluted in appropriate proportions and incubate them with the cells at room temperature. Subsequently, pA/G-Tnp transposons were rotated and incubated with the samples for 1h to activate the translocase fragment DNA and extract DNA.
DNA library was constructed with TruePreP Index Kit V2 for Illumina (TD202, Vazyme Biotech). The library was purified by VAHTS DNA Clean Beads (N411, Vazyme Biotech)

tag: cell line


title: SmoA1_braintumor_IR_5h_repC1

description: 10x Genomics

tag: time


title: Sequencing Lane 2, RNA


title: SmoA1_braintumor_IR_5h_repB2

description: 10x Genomics


title: H3K9ac


title: Control1_1


title: Chip2D1


title: Chip2D2


title: ENL_Y78A


title: H3K9bhb


title: Chip2C12


title: SmoA1_braintumor_IR_24h_repD3

description: 10x Genomics


title: Chip2C11


title: SmoA1_braintumor_IR_5h_repA4

description: 10x Genomics


title: SmoA1_braintumor_IR_24h_repB1

description: 10x Genomics


title: Macrophages-MLKL3

data_processing:
CLC Genomics Workbench v11.0.1
Sequence reads were trimmed for adaptor sequence/low-quality sequence using CLC genomic benchwork (parameter-quality limit:0.05)
Trimmed sequence reads were mapped to GrCM38/mm 10 using CLC genomic benchwork (parameters- mismath cost: 2; insertion cost: 3;  deletion cost: 3;  length fraction: 0.8; similarity faction:0.8)
Read count extraction and normalization were performed using CLC genomic benchwork
Assembly: mm10
Supplementary files format and content: tab-delimited text file includes raw counts of gene matrix for each sample

source_name: PDAC cells

extract_protocol:
RNA  was harvested using Rneasy mini plus kir (Qiagen). 1.3 ug of total RNA was used for the construction of sequencing libraries.
RNA libraries for RNA-seq were prepared using SMARTER mRNA-seq Library Prep Kit following manufacture's protocols.

value: PDAC cells

value: macrophage

value: Co-cultured with PANC1-MLKL


title: Chip2C10


title: SmoA1_braintumor_IR_24h_repA3

description: 10x Genomics

data_processing:
Cell Ranger software v.5.0.1 default pipeline
Assembly: mm10
Supplementary files format and content: Tab-separated values files and matrix files


title: Macrophages-MLKL2


title: Chip2C9


title: ENL_wt


title: Macrophages-MLKL1


title: Capsaicin+Mitoxantrone

city: Iizuka

first: Midori

last: Iida

address: Kawazu 680-4

country: Japan

institute: Kyushu Institute of Technology

description: Gene expression after 24hr incubation with  50 μM of Capsaicin and  30 nM of Mitoxantrone

hyb_protocol: Cy3-labeled cRNA (600 ng, specific activity >6 pmol Cy3/µg cRNA) was fragmented at 60°C for 30 min in a reaction volume of 25 µl containing 25× Agilent fragmentation buffer and 10× Agilent blocking agent following the manufacturer’s instructions. Upon completion of the fragmentation reaction, 25 µl of 2× Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint GE Unrestricted Microarrays (G2519F) for 17 h at 65°C in a rotating Agilent hybridization oven.

scan_protocol: The slides were scanned immediately after washing using an Agilent DNA Microarray Scanner (G2505C) with a one-color scan setting for 8 × 60K array slides (Scan Area 61 × 21.6 mm, Scan resolution 3 µm, Dye channel was set to Green and Green PMT was set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 028282_D_F_20110531) to obtain background subtracted and spatially detrended processed signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.

source_name: expoused to 50 μM of Capsaicin and 30 nM of Mitoxantrone

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from 150 ng RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were assessed using a NanoDrop ND-1000 Spectrophotometer.

growth_protocol: Cells were seeded at a density of 50,000 cells/mL in a 10-cm diameter dish and exposed to various drugs (capsaicin 50 μM, mitoxantrone 30 nM) for 24 h at 37°C in a 5% (v/v) CO2 atmosphere.

extract_protocol: Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol and used for microarray experiments by the well. The extracted total RNA from these wells was combined by exposure condition.

treatment_protocol: Experiments were conducted in three independent wells for each group.


title: Chip2C8

data_processing:
Data quality was assessed with fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Read-trimming was conducted using bbduk (http://jgi.doe.gov/data-and-tools/bb-tools/).
Reads were aligned to the mouse genome using STAR (https://github.com/alexdobin/STAR).
BAM file outputs were mapped to genes using htseq‐count (Anders et al., 2015) using default settings.
Assembly: human genome GRCh38
Supplementary files format and content: .txt files represent number of raw read counts mapping to each gene


title: rbohD_waterlogging_1h_Rep3

city: Columbia

first: Pallav

last: Singh

state: Missouri

address: CE720 Clinical Support and Education

department: Institute for Data Science and Informatics

institute: University of Missouri

data_processing:
Trimgalore 0.6.7 was used for removing Illumina adapters, ambiguous nucleotides (N’s), any sequence reads with total read length < 20 bp and the quality Phred score < 20
Hisat2 version 2.2.1 was used for alinging the sequences with the assembely
Cufflinks version 2.2.1 was used for obtaining the FPKM values as well as cuffdiff method within the package was used for finding out the differentially expressed genes
Assembly: TAIR10
Supplementary files format and content: annotated output with FPKM values in csv file format

organism: Arabidopsis thaliana

source_name: leaves

extract_protocol:
RNA was extracted using Plant RNeasy kit (Qiagen)
RNA libraries were prepared using standard Illumina protocols

value: leaves

value: 60 min

value: rbohD

value: Waterlogging


title: PZH+Anti-pd1, replicate2，

first: Zhiyun

last: Cao

state: 福建Fujian

address: 1# Qiu Yang Road

institute: Fujian university

data_processing:
The sequencing quality were assessed with FastQC (v0.11.5) and then low quality data were filtered using NGSQC (v2.3.3)
The clean reads were then aligned to the reference genome using HISAT2 (v2.1.0)  with default parameters
The processed reads from each sample were aligned using HISAT2 against the reference genome.
The gene expression analyses were performed with StringTie (v1.3.3b). DESeq(v1.28.0) was used to analyze the DEGs between samples.
Assembly: mm 10
Supplementary files format and content: tab-delimited text files including raw counts data for each samples

source_name: Mouse colon

extract_protocol:
Total RNA was extracted with Trizol (Invitrogen) and assessed with Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen),1 μg total RNA meet the following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8.RNA-seq libraries were generated and sequenced
The NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) was used to construct the libraries for sequencing. NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB) kit was used to enrich the poly(A) tailed mRNA molecules from 1 μg total RNA. The mRNA was fragmented into ~200 base pair pieces

value: Mouse colon

value: epithelial cells

value: KRAS and TP53 mutation

value: PZH and Anti-pd1 treatment


title: Chip2C7

extract_protocol:
RNA was acquired through nanoSPLITS. RNA quality and quantity were analysed with a RNA6000 Nano Assay using the Agilent 2100 Bioanalyzer (Agilent Technologies).
For RNA‐Seq, The TruSeq stranded mRNA (cat#20020594) was used to generate cDNA library for illumina NextSeq550 platform according to the manufacture protocol. Single-read sequencing of the cDNA libraries with a read length of 150 was performed with NextSeq 500 Sequencing System using NextSeq 500/550 High Output v2 kit 150 cycles (cat#20024907).

value: Human pancreatic islet cell

value: primary cells from 2 human donors


title: PZH+Anti-pd1, replicate1，


title: Mitoxantrone only

description: Gene expression after 24hr incubation with 30 nM of Mitoxantrone

source_name: expoused to 30 nM of Mitoxantrone


title: Serous cystic neoplasm of the pancreas tissue_4

city: Tokyo

first: Nobuyoshi

last: Hiraoka

address: 5-1-1 Tsukiji, Chuo-ku

department: Pathology Division

institute: National Cancer Center Research Institute

description: Gene expression

hyb_protocol: Hybridization was performed using a Gene Expression Hybridization kit (Agilent Technologies) according to manufacturer's instruction.

scan_protocol: The slide was scanned with an Agilent Technologies Scanner G4900DA.

data_processing: Agilent Feature Extraction Software (v12.1.1.1) was used for background subtraction.

label_protocol: Cyanine-3 (Cy3) labeled cRNA was prepared from the RNA using the Low Input Quick Amp LabellingKit One colour (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's instructions. Dye incorporation and cDNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.

extract_protocol: Total RNA was extracted from the tissues using an RNeasy-Mini kit (QIAGEN, Valencia, CA) and quantified using a NanoDrop 8000 instrument (Thermo Fisher Scientific, Waltham, MA). The RNA integrity number (RIN) was evaluated by a 2100 Bioanalyzer (Agilent, Santa Clara, CA).

value: Serous cystic neoplasm

tag: gender

value: female

tag: age


title: rbohD_waterlogging_1h_Rep2


title: Macrophages-Vec2

value: Co-cultured with PANC1-Vector


title: Control

description: Gene expression after 24hr incubation

source_name: control (nothing expoused)


title: Capsaicin only

description: Gene expression after 24hr incubation with  50 μM of Capsaicin

source_name: expoused to 50 μM of Capsaicin


title: Serous cystic neoplasm of the pancreas tissue_3

description: Gene expression


title: Chip2C6

source_name: Human pancreatic islet cell

tag: medium

value: pancreatic islet media (PIMS, Prodo Laboratories)

tag: time point

value: 24 hour culture


title: Serous cystic neoplasm of the pancreas tissue_5

description: Gene expression


title: rbohD_waterlogging_1h_Rep1


title: Macrophages-Vec3

value: PDAC cells

value: macrophage


title: Anti-pd1, replicate3，

value: Anti-pd1 treatment


title: Anti-pd1, replicate2，


title: Chip2C5


title: Serous cystic neoplasm of the pancreas tissue_2

description: Gene expression

value: male


title: Serous cystic neoplasm of the pancreas tissue_1

description: Gene expression


title: Chip2C4


title: Anti-pd1, replicate1，


title: PZH+Anti-pd1, replicate3，


title: Chip2C3


title: rbohD_control_1h_Rep2

value: leaves

value: 60 min

value: rbohD


title: PZH, replicate3，

value: Mouse colon

value: epithelial cells

value: PZH treatment


title: Patient 5

city: St. Louis

first: Jeffrey

middle: Robert

last: Millman

state: MO

address: 660 Euclid

institute: Washington University in St. Louis

description:
Patient 5 Fixed DMSO
Patient 5 Fixed BFA
Patient 5 Fixed TG
Patient 5 Fixed CM
Patient 5 Fixed IL1BIFNG
Patient 5 Fixed IL1B
Patient 5 Fixed IFNG
Patient 5 Fixed TNFA

data_processing:
Data was generated and processed by McDonnell Genome Institute (MGI) at Washington University
Assembly: GRCh38-2020-A
Supplementary files format and content: barcodes.tsv, features.tsv, matrix.mtx

source_name: Pancreatic Islet

growth_protocol: CMRL+10% FBS

extract_protocol:
Islets were single cell dispearsed and 10x fixed single-cell RNA sequencing protocol was used.
Library construction for single cell was constructed using 10X Chromium Protocol.

treatment_protocol: scRNA-seq islets were treated for 48-hr with 1,000 ng/mL IFNγ + 500 ng/mL TNFα+100 ng/mL IL1β ; 1,000 ng/mL IFNγ+100 ng/mL IL1β; 100 ng/mL IL1β ; 1,000 ng/mL IFNγ; 500 ng/mL TNFα ; 10 μM thapsigargin (TG), or 1 ug/mL brefeldin A (BFA) or DMSO

value: Pancreatic Islet

value: Islets of Langerhans

value: DMSO, BFA, TG, CM, IL1BIFNG, IL1B, IFNG, or TNFA


title: Macrophages-Vec1


title: rbohD_control_1h_Rep1


title: Patient 4

description:
Patient 4 Fixed DMSO
Patient 4 Fixed BFA
Patient 4 Fixed TG
Patient 4 Fixed CM
Patient 4 Fixed IL1BIFNG
Patient 4 Fixed IL1B
Patient 4 Fixed IFNG
Patient 4 Fixed TNFA


title: PANC-1 cells, FBXO32 depeleted, bioreplicate 2

city: Beijing

first: Dan

last: Su

address: No. 1 Shuaifuyuan, Wangfujing Street, Beijing

department: Department of General Surgery

institute: Peking Union Medical college hospital

description: ribosome-protected fragments (RPFs)

data_processing:
Sequencing reads from Ribo-seq were first filtered by fastp to remove low-quality reads, and then filtered using the short reads alignment tool Bowtie2 (version 2.2.8) to further remove reads that were mapped to ribosome RNA (rRNA), transfer RNAs (tRNA), small nuclear RNAs (snRNA), small nucleolar RNAs (snoRNA), and miRNA.
The sequencing reads were mapped to human genome by STAR software and then quantified using the RSEM software by calculating the TPM (Transcript per Kilobase per Million mapped reads) values.
Differential expression analysis was performed using DESeq2 software
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files includes TPM values of each sample

growth_protocol: PDAC cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM, Viacell) which were supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% normocin (Invivogen), at 37 °C in a humidified incubator containing 5% CO2

extract_protocol:
PANC-1 cells were incubated with 100 μg/mL CHX at 37 °C for 2 min to terminate the movement of ribosomes along mRNAs. Cells were then wash twice with ice-cold PBS containing 100 μg/mL CHX to remove dead cells and cell debris, and scratched off in ice-cold PBS containing 100 μg/mL CHX to collect PDAC cells. After centrifugation, the cell pellets were resuspended in polysome lysis buffer (20 mM HEPES (pH 7.4), 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 μg/mL cycloheximide), incubated on ice for 10 min, triturated ten times through a 26-G needle, and centrifuged at 20000g for 10min at 4℃ to collect supernatants. To prepare ribosome-protected fragments (RPFs), RNase I (NEB, Ipswich, MA, USA) and DNase I (NEB, Ipswich, MA, USA) were added into cell lysates to digest the free RNAs and the nuclease digestion reaction was kept for 45 min at RT with gentle mixing. Isolation of RPFs with a size greater than 17nt was achieved by size-exclusion chromatography with MicroSpin S-400 HR columns (GE Healthcare, USA) and RNA Clean and Concentrator-25 kit (Zymo Research; R1017) following the instructions of the manufacturer.
After removing contaminating rRNA, the recovered RPFs were then applied for library construction using NEBNext® Multiple Small RNA Library Prep Set for Illumina®

treatment_protocol: PANC-1 cells  grown in 15-cm culture dishes were collected for ribosome profiling when the confluency reached 80% to 90%.

value: pancreatic adenocarcinoma cells


title: PZH, replicate2，

value: KRAS and TP53 mutation


title: Chip2C2

first: James

last: Fulcher

institute: Pacific Northwest National Laboratory

value: Human pancreatic islet cell

value: primary cells from 2 human donors

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: rbohD_0min_Rep3

value: 0 min


title: Chip2C1

value: 24 hour culture


title: PANC-1 cells, FBXO32 depeleted, bioreplicate 1

description: ribosome-protected fragments (RPFs)


title: PANC-1 cells, negative control, bioreplicate 1

description: ribosome-protected fragments (RPFs)


title: Chip2B12


title: rbohD_control_1h_Rep3


title: rbohD_0min_Rep2


title: PBS, replicate3，

value: PBS as control


title: PANC-1 cells, negative control, bioreplicate 2

description: ribosome-protected fragments (RPFs)


title: Chip2B11


title: PBS, replicate2，


title: WT_waterlogging_1h_Rep3

value: leaves

value: Waterlogging


title: Chip2B10


title: rbohD_0min_Rep1

value: rbohD


title: DOXY+_ARS-_FUS-P525L_CLIP_REP2

city: Rome

first: Irene

last: Bozzoni

address: Piazzale Aldo Moro 5

department: Biology and Biotechnologies "Charles Darwin"

country: Italy

institute: Sapienza University of Rome

description:
IIT_FCP_IP_6_S34
SK-N-BE stable cell line with doxycyclin inducible FUS-P525L,CLIP (with doxycyclin induction), Replicate2
omniclip_peaks.bed

data_processing:
*library strategy: CLIP-seq
Trimmomatic (v0.39; Bolger et al., 2014) and Cutadapt (v3.2; Martin, 2011) were used to remove adapter sequences and poor quality bases; minimum read length after trimming was set to 35.
Reads aligning to rRNAs, 7SL or 7SK RNAs were filtered out; this first alignment was performed using Bowtie2 software (v2.4.2, https://bowtie-bio.sourceforge.net/bowtie2/index.shtml).
STAR software (v2.7.7a, Dolbin et al., 2013) was used to align reads to GRCh38 genome with these parameters: --readFilesCommand zcat --outSAMtype BAM Unsorted  --alignEndsType EndToEnd --outFilterMultimapNmax 10 --outFilterIntronMotifs RemoveNoncanonical --alignSJoverhangMin 12 --outFilterMatchNmin 15 --outFilterMismatchNoverLmax 0.05 --outFilterMultimapScoreRange 3 --alignIntronMax 20000 --seedMultimapNmax 200000 --seedPerReadNmax 30000 --peOverlapNbasesMin 35 --outSAMattributes NH HI NM MD AS nM jM jI XS
PCR duplicates were removed from all samples using MarkDuplicates command from Picard suite (v2.24.1; https://broadinstitute.github.io/picard/).
Peak calling was performed using omniCLIP software selecting only significant peaks (bonferroni corrected p-value < 0.05) (https://github.com/philippdre/omniCLIP?tab=readme-ov-file#usage). Bigwig files related to reads coverage in each sample were generated using bamCoverageRead1tion of defor each strand  ()c
Assembly: GRCh38
Supplementary files format and content: omniclip_peaks.bed contains FUS-P525L RNA binding sites
Supplementary files format and content: bigwig files related to reads coverage in each sample

source_name: SK-N-BE

growth_protocol: SK-N-BE cells were cultured in RPMI (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% FBS (Sigma-Aldrich, #F2442), GlutaMAX supplement 1X (ThermoFisher Scientific, # 35050061), sodium-pyruvate 1mM (Thermo Fisher Scientific, #11360070) and Pen/Strep 1X (Sigma-Aldrich, #P4458).

extract_protocol:
RNA was extracted with TRIazol reagent according to the manufacturer’s specifications.
RNA libraries for RNA-seq were prepared using Stranded Total RNA Prep with Ribo-Zero Plus (Illumina) following manufacturer's protocols.

treatment_protocol: FUS HITS-CLIP was performed according to the protocol described by Huppertz et al. (2014) with some adjustments. In particular, 10 milions SK-N-BE cells were exposed to 50 ng/mL Doxycycline (Sigma-Aldrich, #D9891) for 24h. Cells were UV-C crosslinked and lysed. The extract was treated with RNase I (1:200) and Turbo DNase for 3 min at 37°C. 2-3 mg of extract were diluted 2mg/ml with lysis buffer and incubated with 100 ml of Anti- flag beads (Sigma, # M8823) for 2hr at 4°C. Beads were washed according to Huppertz et al. and resuspended in 190 ml of PK buffer added with 10ml of PNK and incubated for 20min shaking at 1100 rpm at 37°C. After this step, 200 ml of PK buffer + 7M urea were added and incubated for 20min at 1100 rpm at 37°C.

value: SK-N-BE

value: Neuroblastoma

value: Doxycyxlin


title: PBS, replicate1，

value: Mouse colon

value: epithelial cells


title: DOXY+_ARS-_FUS-P525L_CLIP_REP1

description:
IIT_FCP_IP_3_S29
SK-N-BE stable cell line with doxycyclin inducible FUS-P525L,CLIP (with doxycyclin induction), Replicate1
omniclip_peaks.bed


title: Chip2B9

address: 902 Battelle Blvd


title: WT_waterlogging_1h_Rep2

value: 60 min


title: Aged mice TEM, rep 2

city: Memphis

first: Tian

last: Mi

address: 262 Danny Thomas Pl MS351

department: Immunology

institute: St. Jude Children's Research Hospital

description:
WGBS
2-2128829

data_processing:
Basecalling was performed using Illumina Casava 1.7
Sequencing reads were aligned to the mm10 genome using the BSMAP v.2.90 software
CpG methylation levels were determined by methratio.py script from BSMAP
Assembly: mm10
Supplementary files format and content: txt files with columns of chr, loc, cov, meth

source_name: blood

extract_protocol:
DNA was extracted from the sorted cells and then bisulfite treated using an EZ DNA Methylation-Direct kit (Zymo Research).
Bisulfite-modified DNA sequencing libraries were generated using the EpiGenome kit (Epicentre) according to the manufacturer’s instructions. Bisulfite-modified DNA libraries were sequenced using Illumina NovaSeq 6000 systems

value: blood

value: CD8 T cells


title: DOXY+_ARS-_FUS-P525L_INP_REP2

description:
IIT_FCP_INPUT_6_S30
SK-N-BE stable cell line with doxycyclin inducible FUS-P525L,INPUT (with doxycyclin induction), Replicate2
omniclip_peaks.bed


title: PZH, replicate1，

value: KRAS and TP53 mutation

value: PZH treatment


title: Chip2B8

value: Human pancreatic islet cell

value: primary cells from 2 human donors

value: pancreatic islet media (PIMS, Prodo Laboratories)


title: WT_control_1h_Rep3


title: Aged mice TCM, rep 2

description:
WGBS
2-2128828


title: DOXY+_ARS-_FUS-P525L_INP_REP1

description:
IIT_FCP_INPUT_3_S10
SK-N-BE stable cell line with doxycyclin inducible FUS-P525L,INPUT (with doxycyclin induction), Replicate1
omniclip_peaks.bed


title: Aged mice Naive, rep 2

description:
WGBS
2-2128827


title: Chip2B6

value: 24 hour culture


title: scRNA-seq of hiPSC-derived striato-nigral circuitoids: 80 days in vitro, Library2

city: Nanjing

first: Yan

last: Liu

state: Jiangsu

address: 101Longmian Avenue, Jiangning District

department: School of Pharmacy

institute: Nanjing Medical University

data_processing:
For the scRNA-seq, raw sequencing data was processed with Cell Ranger software version 3.1 (10X Genomics) for FASTQ generation and GRCh38 human reference genome was used for alignments. For filtering and unsupervised clustering, the gene expression matrixs from Cell Ranger results were used for downstream analysis, using R 4.1.1.
Assembly: hg38
Supplementary files format and content: outputs of cellranger software, which include barcodes, gene features and expression matrix for each sample

source_name: brain organoids

extract_protocol:
For the scRNA-seq, brain organoids were dissociated by 1 ml of trypLE (Life Technology Inc) for 30 min in the condition of 37°C and 5% CO2 into single cell when differentiated in vitro for 35 days. The cell debris and other aggregates were removed by additional low-speed centrifugation. Droplet-based single-cell RNA sequencing was performed using the 10X Genomics Chromium Single Cell platform, version 3. Approximately 5,000 cells were captured for each sample onto a Chromium Single Cell 3’ Chip (10X Genomics, PN-120236), and the released RNA in each cell was barcoded through reverse transcription in individual gel beads in emulsions (GEMs).
For the scRNA-seq, S1000TM Touch Thermal Cycler (Bio Rad) was used for reverse transcription at 53°C  for 45 min, followed by 85°C for 5 min and final hold at 4°C. The quality of cDNA was accessed on Agilent 4200 (Agilent Technologies). Libraries of scRNA-seq were established by the Chromium Single Cell 3’ Library & Gel Bead Kit V3 (10X Genomics, 1000075) and then sequenced on an Illumina NovaSeq6000 with a sequencing depth of at least 750000 reads per cell.

value: brain organoids

value: neural cells


title: scRNA-seq of hiPSC-derived striato-nigral circuitoids: 80 days in vitro, Library1


title: Aged mice TEM, rep 1

description:
WGBS
2-2128826


title: Chip2B5

city: Richland


title: WT_waterlogging_30min_Rep3

value: leaves

value: 30 min

value: Waterlogging


title: scRNA-seq of hiPSC-derived striatal organoids: 80 days in vitro, Library2


title: Chip2B4


title: Aged mice Naive, rep 1

description:
WGBS
2-2128824


title: Chip2B3


title: WT_control_1h_Rep1


title: Half Lifetime, rep 3, 450 days

description:
WGBS
2-1849782

value: multi-lifetime iteratively stimulation


title: WT_waterlogging_30min_Rep1


title: Aged mice TCM, rep 1

description:
WGBS
2-2128825


title: WT_waterlogging_30min_Rep2


title: Chip2B2


title: WT_waterlogging_1h_Rep1


title: Half Lifetime, rep 2, 450 days

description:
WGBS
2-1849781


title: treatment-3

first: Chengyu

last: Liao

address: 134, east road

institute: Fujian Medical University

description: co-CM-MLKL-treated panc1

data_processing:
none provided
Supplementary files format and content: counts

extract_protocol: none provided

value: PDAC cells

value: cancer cells

value: wild-type

value: Co-cultured CM-MLKL


title: scRNA-seq of hiPSC-derived striatal organoids: 80 days in vitro, Library1


title: WT_control_30min_Rep3


title: WT_control_1h_Rep2

value: 60 min


title: Chip2B1


title: Half Lifetime, rep 1, 450 days

description:
WGBS
2-1849780

value: blood

value: CD8 T cells


title: treatment-1

description: co-CM-MLKL-treated panc1


title: Chip2A12

value: Human pancreatic islet cell


title: treatment-2

description: co-CM-MLKL-treated panc1


title: Chip2B7

value: primary cells from 2 human donors


title: WT_control_30min_Rep1


title: control-3

description: co-CM-Vector-treated panc1

value: Co-cultured CM-Vector


title: 2 Lifetime, rep 5, 1620 days

description:
WGBS
2-1849779


title: 4 Lifetime, rep 3, 2880 days

description:
WGBS
2-1748680


title: 2 Lifetime, rep 4, 1620 days

description:
WGBS
2-1849778


title: WT_waterlogging_10min_Rep3

value: 10 min


title: control-2

description: co-CM-Vector-treated panc1


title: WT_waterlogging_10min_Rep2


title: Chip2A9

value: pancreatic islet media (PIMS, Prodo Laboratories)

value: 24 hour culture


title: WT_waterlogging_10min_Rep1

value: leaves


title: Chip2A11


title: Chip2A10


title: 2 Lifetime, rep 3, 1620 days

description:
WGBS
2-1849777


title: control-1

description: co-CM-Vector-treated panc1


title: HDF cells, DHACM +TGFβ1 treated, rep 2

city: Marietta

first: Isioma

last: Enwerem-Lackland

state: GA

address: 1775 W Oak Commons Ct

department: Research and Development

institute: Mimedx

data_processing:
Raw data (raw reads) of fastq format were firstly processed through in-house (Novogene) perl scripts
Paired-end reads were aligned to the reference genome using Hisat2 v2.0.5.
FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene
Differential expression analysis  was performed using the DESeq2 R package (1.20.0)
Assembly: hg38
Supplementary files format and content: excel file includes FPKM values for each sample

source_name: HDF

growth_protocol: Adult human dermal fibroblast cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humdified atmosphere with 5% CO2 at 37C

extract_protocol:
RNA extraction was done at Novogene  using TRIzol following manufacturer's protocols
mRNA library preparation was done at Novogene

treatment_protocol: To initiate experiments under MMC conditions, human dermal fibroblast (HDF) cells were seeded in 100mm dishes at 10,000 cells/cm2 for each experiment. After 4 hours, cells were washed with basal DMEM, and starved overnight in DMEM containing 0.4% FBS, 1% penicillin streptomycin, and 1% sodium pyruvate followed by treatment of HDFs for six days. Culture media and treatments were replaced on day 3.  All treatments were carried out in DMEM containing 0.4% FBS, 1% penicillin streptomycin, 1% sodium pyruvate, 1 mM of L-ascorbic acid 2-phospate (Sigma-Aldrich, St. Louis, MO), and a mixture of 37.5 mg/mL Ficoll 70 kDa (Sigma-Aldrich, St. Louis, MO) and 25 mg/ml Ficoll 400 kDa (Sigma-Aldrich, St. Louis, MO). The following control groups were included for each experiment: Basal media and 10 ng/mL TGFβ1 (R&D Systems, Minneapolis, MN). Treatments were carried out with DHACM (20 mg/mL, 10 mg/mL, and 1 mg/mL) in the presence of 10 ng/mL TGFβ1. Extracts were prepared as described in Moreno et al, 2021.

value: HDF

value: Human dermal fibroblasts

value: WT

value: macromolecular crowding model + DHACM  + TGFβ1


title: WT_control_10min_Rep3


title: Chip2A8


title: Chip2A7


title: 4 Lifetime, rep 1, 2880 days

description:
WGBS
2-1748677

value: multi-lifetime iteratively stimulation


title: HDF cells, DHACM +TGFβ1 treated, rep 3


title: 4 Lifetime, rep 2, 2880 days

description:
WGBS
2-1748678


title: WT_control_30min_Rep2

value: 30 min


title: WT_control_10min_Rep2


title: Chip2A6


title: WT_control_10min_Rep1


title: HDF cells, DHACM +TGFβ1 treated, rep 1


title: Endogenous, rep 2, 180 days

description:
WGBS
2-1748676


title: Chip2A5


title: Endogenous, rep 1, 180 days

description:
WGBS
2-1748675


title: Chip2A4


title: HDF cells,TGFβ1 treated, rep 2

value: macromolecular crowding model + TGFβ1


title: HDF cells, DHACM treated, rep 3

value: macromolecular crowding model + DHACM


title: Chip2A2


title: Chip2A3


title: 2 Lifetime, rep 1, 1350 days

description:
WGBS
2-1748672

value: blood


title: HDF cells,TGFβ1 treated, rep 1


title: WT_0min_Rep1

value: 0 min


title: ILC-enriched lung, Alt.BPC, scRNA-seq

first: Yung-An

last: Huang

address: 9500 Gilman Dr

department: Medicine

institute: University of California, San Diego

description: Fluent BioSciences

data_processing:
The demultiplexing was completed by the outsourced sequencing core, while barcode processing, gene counting, and aggregation were performed using the PIPseeker software (02.00.00).
Assembly: GRCm39
Supplementary files format and content: Tab-separated values files and matrix files

source_name: ILC-enriched lung

extract_protocol:
The lungs were collected in free RPMI medium and then dissociated for single-cell suspension by the Miltenyi Lung Digest Kit and Dissociator. ILCs from Alt and Alt plus BPC treated mice were enriched from two single-cell suspended lungs by Mouse Pan-ILC Enrichment Kit. ILC enriched samples were preserved in PIPseqTM T100 3ʹ Single Cell Capture and Lysis Kit and processed by sequencing service.
Library was performed according to manufacter's instructions (FB0003657 PIPseq T100 v3.0 Single Cell RNA Kit User Guide Rev 1.4, Fluent BioSciences).

value: ILC-enriched lung

value: Alternaria alternata+BPC


title: HDF cells, DHACM treated, rep 1


title: HDF cells, DHACM treated, rep 2


title: ILC-enriched lung, Alt, scRNA-seq

description: Fluent BioSciences

value: Alternaria alternata


title: WT_0min_Rep2


title: S1-seq_wt_timecourse_16dpp_rep3

first: Soonjoung

last: Kim

address: 430 East 67th Street

institute: Memorial Sloan Kettering Cancer C

data_processing:
After Qubit quantification and quality control by Agilent BioAnalyzer, libraries were pooled equimolar and run on an Illumina sequencer. We obtained paired-end reads of 50 bp. A spike-in of PhiX was added to the run to increase diversity when necessary and for quality control purposes. Base calls were perfomed using bcl2fastq software v.2.20 or bcl-convert software v.3.9.3 to 3.10.5 for samples sequenced on Illumina NextSeq 500, NextSeq 2000, or HiSeq 4000.  Reads were trimmed and filtered by Trim Galore version 0.6.6 with the arguments --paired --length 15 <http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/>. Sequence reads were mapped onto the mouse reference genome (mm10) by bowtie2 version 2.3.5.1 (ref. 1) with the arguments -N 1 -X 1000.  (ref. 1) Langmead et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10, R25 (2009).
Duplicated reads were removed by Picard <https://broadinstitute.github.io/picard/>. Uniquely and properly mapped reads (MAPQ ≥ 20) were extracted by samtools with the arguments -q 20 <http://www.htslib.org/>.
Reads for S1, Exo7/T, or ExoT-seq were counted at which a nucleotide next to biotinylated adaptor DNA was mapped (this corresponds to a position of ssDNA). Maps were analyzed using R version 4.0.3 <http://www.r-project.org>.
Assembly: mm10
Supplementary files format and content: BigWig files contain read counts at positions of resection endpoints. Reads were not normalized to RPM (reads per million mapped). Letter "F" or "R" in sample names indicates that reads were mapped on forward or reverse strand, respectively.

source_name: testis

extract_protocol:
Testis cells from mice at indicated age were obtained as described previously (ref. 1). Cells were embedded in plugs of low-melting point agarose to protect DNA from shearing, and DNA was liberated by treatment with SDS and proteinase K as described previously (ref. 1).  (ref. 1) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020).
In-plug overhang removal with S1 endonuclease for S1-seq, exonuclease VII and exonuclease T for Exo7/T-seq, or exonuclease T for ExoT-seq, and adaptor ligation were performed as described previously (ref. 1, 2, 3) with minor modifications. In brief for S1-seq, plugs were washed in 0.5 ml of modified S1 buffer (50 mM sodium acetate, 280 mM NaCl, 4.5 mM ZnSO4, pH 4.7) for 30 min four times, treated with 9 units of S1 nulcease in 500 µl of modified S1 buffer at 37°C for 20 min and washed once with 1ml TE. For Exo7/T-seq, plugs were washed in 1 ml of exonuclease VII buffer for 15 min twice, treated with 50 units of exonuclease VII in 100 µl of exonuclease VII buffer at 37°C for 60 min and washed once with 1ml TE and followed by exonulcease T treamtment. For ExoT-seq, plugs were washed in 1 ml of NEBuffer 4 for 15 min 3 times, treated with 75 units of exonuclease T in 100 µl of NEBuffer 4 at 24°C for 90 min and washed 3 times with 8 ml of TE for 15 min. The nuclease treated ssDNA ends were further polished with T4 polymerase before the first adaptor ligation as previously described (ref. 1, 2, 3). After ligation to biotinylated adaptors, DNA was purified from the agarose, sheared by sonication, purified with streptavidin, ligated to second-end adaptors, amplified, and sequenced as described previously (ref. 1, 2, 3).  (ref. 1) Yamada et al., Molecular structures and mechanisms of DNA break processing in mouse meiosis. Genes Dev 34, 806-818 (2020).  (ref. 2) Mimitou et al., S. A global view of meiotic double-strand break end resection. Science 355, 40-45 (2017).  (ref. 3) Mimitou et al., S1-seq Assay for Mapping Processed DNA Ends. Methods Enzymol 601, 309-330 (2018).
S1-seq, Exo7/T-seq, ExoT-seq

value: testis

tag: strain

value: Spermatocytes

value: wild type

value: 16 days post partum


title: HDF cells,TGFβ1 treated, rep 3

value: HDF


title: S1-seq_Rad50DY_14dpp_rep1

value: 14 days post partum


title: Egr1 KO_PBS_2

city: Rehovot

first: Lingling

last: Zhang

address: Yavne Street 68, 68

department: Molecular Cell Biology

country: Israel

institute: Weizmann Institute of Science

description: 10x Genomics

data_processing:
The demultiplexing, barcoded processing and gene counting were made using the Cell Ragner software v7
Assembly: mm10
Supplementary files format and content: tar archive of tab-separated values files and matrix files from cellranger

source_name: heart

extract_protocol:
Single-cell preparation for single-cell RNA sequencing 1. Cardiac cells were isolated from mouse hearts 4 days after myocardial infarction using a neonatal heart dissociation kit Primary materials needed: Enzyme mixtures (Miltenyi Biotec, Cat# 130-098-373), surgery tools, butterfly (KRUUSE Butterfly, Cat# 121716), GentleMACS dissociator (Miltenyi Biotec, Cat# 130-093-235), ice-cold PBS, 10cm dish, 6-well plate, GentleMACS C-tube (Miltenyi Biotec, Cat# 130-093-237), 10cm injector, 70 μm strainer, 50ml tube, wide-bore tips. 1). Euthanize mice using CO2. 2). Perfuse the heart with 20 mL of ice-cold PBS. 3). Identify and cut the infarcted area, mainly the left ventricle. 4). Wash the tissue twice in ice-cold PBS, ensuring complete blood removal with tweezers. 5). Transfer the tissue to a 6-well plate with 2 ml of enzyme mixture. Chop the tissue into fine pieces using scissors and transfer each piece to a separate C-tube. Run the dissociator program once (gently shake if some tissue is stuck in the middle). 6). Follow the GentleMACS incubation protocol (15 min each, 3-4 times). 7). After incubation, spin down for a few seconds to let all the mixture settle at the C-tube bottom. Add 5 mL of medium to each C-tube and centrifuge at 1400rpm for 5 minutes at 4°C. 8). Remove the supernatant completely and suspend the pellet in 3.1 ml of ice-cold PBS for each C-tube, using 1 ml wide-bore tips. 9). Run the suspension through a 70 μm strainer, with a 15ml tube as a container. 2. Debris removal Primary materials needed: ice-cold PBS, Debris removal solution (Miltenyi Biotec, Cat# 130-109-398) 1). Add 900 μl of Debris Removal solution to each C tube. 2). Mix well by gently moving the pipette 10 times up and down using wide-bore tips. 3). Overlay very gently with 4 ml of ice-cold PBS. (Note: Tilt the tube and pipette very slowly to ensure the newly added PBS is not mixed with the cell suspension.) 4). Centrifuge at 1000g for 10 minutes at 4°C with full acceleration and full brake. Three phases will be formed. 5). Aspirate the two top phases thoroughly and gently using a vacuum and a 1 ml pipette. 6). Fill up with cold PBS to the final 15 ml. 7). Gently invert the tube three times. Do not vortex. 8). Centrifuge at 1000g for 10 minutes at 4°C. 9). Aspirate the supernatant completely. 3. Red Blood Cell Lysis Primary materials needed: MACS buffer (0.5% BSA, 2mM EDTA in PBS, pH 7.2) at room temperature (RT), RBC lysis buffer (Miltenyi Biotec, Cat# 130-094-183) at RT. 1). Resuspend the cell pellet in 1 ml MACS buffer at RT using wide-bore tips. 2). Add 10 mL of RBC lysis buffer at RT. Gently pipette up and down 10 times. 3). Incubate at room temperature for 2 minutes. 4). Centrifuge at 600g for 5 minutes at 4°C. Pipette off the supernatant completely. 4. Magnetic Labeling Primary materials needed: ice-cold MACS buffer, CD31 microbeads (Miltenyi Biotec, Cat# 130-097-418), CD45 microbeads (Miltenyi Biotec, Cat# 130-052-301), wide-bore tips (200um, 1ml), Maintain room temperature at 20 degrees Celsius (lower air conditioner). 1). Take one aliquot for calibration by FACS to determine the percentage of all cell types. 2). Mix microbeads before use. Incubate cell pellets with microbeads: 10ul CD45 + 10ul CD31 microbeads + 70ul MACS buffer. 3). Mix well and incubate microbeads for 15 minutes at 4-8 degrees (refrigerator, not on ice). Gently mix once every 5 minutes. Note: Working on ice may require increased incubation times. Higher temperatures or longer incubation times lead to non-specific cell labeling. 4). Add 1 mL of cold MACS buffer and centrifuge at 300g for 5 minutes at 4°C. 5). Remove the supernatant. Resuspend in 500 uL cold MACS buffer (Up to 108 total cells). Note: Work quickly, maintain cell coldness, and use pre-cooled solutions to prevent antibody capping on the cell surface and non-specific cell labeling. The volumes provided for magnetic labeling are for up to 107 total cells. For fewer than 107 cells, use the same volumes as indicated. Scale up all reagents and total volumes for higher cell numbers. 5. Magnetic Separation - This section is conducted in a cold room (4°C). Primary materials needed: MS column, OctoMACS separator (Miltenyi Biotec, Cat# 130-042-109), MACS buffer, Maintain a lower AC temperature (20°C). 1). Prepare the MS Column by rinsing with buffer: apply 500 μL of MACS buffer on top of the column and allow the buffer to run through. MS Columns are "flow stop," and should not run dry. (Filling with 500 μL takes ~1.5 minutes.) 2). Discard the effluent and change the collection tube. The MS Column is now ready for magnetic separation. 3). Apply the cell suspension onto the prepared MS Column. Collect the flow-through containing unlabeled cells. 4). Wash the MS column with 3X 500 μl of MACS buffer. Collect unlabeled cells that pass through and combine with the flow-through from the last step. (1st: ~1.5 min; 2nd: ~2 min; 3rd: ~3 min) Note: Perform washing steps by adding buffer aliquots as soon as the column reservoir is empty. 5). Remove the MS Column from the separator and place it on a new collection tube. 6). Pipette 1 mL of buffer onto the MS Column. Immediately flush out the fraction with the magnetically labeled cells by firmly applying the plunger supplied with the column. 7). Centrifuge the collected flow-through depletion cells (mainly cardiac fibroblasts)  and beads enriched cells (mostly immune cells) at 300g for 5 minutes at 4°C. 8). Resuspend the cells in 50-500 μl (adjusting based on the cell pellet size) ) of Cell Resuspension Buffer containing 0.04% BSA in PBS, pH 7.2. Count the cells and adjust the concentration to approximately 1X10⁶ cells/mL. 9). Combine 45 μl of depletion cells (~1X10⁶ cells/mL) with 15 μl of enriched cells (~1X10⁶ cells/mL) in one Eppendorf tube. Bring the samples for processing immediately. Note: Use the column immediately after filling to prevent the formation of air bubbles caused by warming up. Do not store columns after filling. The time required to fill the column with buffer depends on storage conditions, temperature, and humidity. Therefore, the filling time may vary from a few seconds to several minutes. This filling time does not influence the quality of the separation.
Library preparation and sequencing of scRNA-seq scRNA-seq was performed using the Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3, 16 rxns (10x Genomics, PN-1000075) according to the manufacturer’s instructions and targeting a recovery of 6,000 cells per dataset. Each dataset was obtained with a sample size of three mouse biological replicates. The libraries were constructed as instructed in the manufacturer’s protocol. Sequencing was done on a Nova-Seq6000 using SP, 100 cycles kit mode allocating 800M reads in total (Illumina).The average read depth across the samples was 23,211 per cell.

value: heart

value: PBS